Two NF membranes were applied to separate and purify the soybean oligosaccharide (SBOS) from SBOS broth. NF-3A membrane was more appropriate than NF-2A membrane because of its superior separation performance. Constant volume diafiltration mode was used to enhance the purity of SBOS. A difference in the rejection and the yield between the SBOS and other sugars ensured the purity enhancement. Under optimal operation conditions, the yield and the purity of the SBOS could reach up to 83.2% and 77.9%, respectively. Deteriorated membrane performance resulting from the membrane fouling could be recovered via specific chemical cleaning process. NF technology with high-efficiency and eco-friendly properties demonstrates the great potential for SBOS purification in practical application.